Project Summary: The parasite Toxoplasma gondii is a serious pathogen of humans and livestock worldwide. T. gondii causes abnormal fetal development or death in a developing fetus, and encephalitis in immunocompromised patients. The ultimate goal of this project is to develop a vaccine able to protect against T. gondii infection. A key concern with any live T. gondii vaccine is that the strain be unable to form persistent cysts, the hallmark of chronic infection. Conversion from the fast growing stage to the encysted stage is critical for persistence within the host, though little is known about this interconversion. In this project, genes involved in cyst formation will be characterized, and an encystation mutant will be identified as a vaccine strain candidate. A library of T. gondii insertional mutants was screened in vitro and in vivo for encystation defects. Nine mutants identified from these screens are the focus of this application. These mutants will be analyzed quantitatively for cyst formation in vivo. The insertion site of the mutagenizing plasmid has been identified for all but one mutant. The insertion site will be determined for this remaining mutant, and the disrupted genes will be confirmed by northern hybridization. Mutants will be complemented by resupply of intact copies of the disrupted genes, demonstrating that these loci are involved in cyst formation. Select mutants will be tested for their ability to maintain a persistent infection in mice. Antisera generated against the identified cyst development factors will be used in expression and localization studies to characterize these proteins. Strains unable to maintain cysts in mice will be checked for their ability to protect mice from lethal infection of T. gondii. Relevance: Our research focuses on identifying factors important in the development of the slow-growing, persistent form of T. gondii, and generating a strain of the parasite unable to transition to this form or remain in the host long-term. This strain will be tested as a vaccine to help the immune system clear infecting parasites before allowing them to convert to the persistent form.